Cardiac Sarcomere Protein Quality Control in Health and Disease

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Cardiomyocytes are essentially non-renewing, so proteotoxicity from dysregulated Protein Quality Control (PQC) is intimately associated with heart failure. The proteins that comprise the cardiac sarcomere are responsible for force generation in the myocyte, and this constant mechanical stress uniquely predisposes them to misfolding. Despite PQC’s central role in heart failure, and the particular vulnerability of the sarcomere to misfolding, the PQC mechanisms that maintain the cardiac sarcomere are almost entirely unknown. This is a critical knowledge gap that we will address in this proposal. Our past work described a z-disc localized complex, anchored by the co- chaperone Bcl2-associated athanogene-3 (BAG3), that was essential for sarcomere PQC. Z-disc BAG3 levels were depressed in human heart failure (HF) and correlated with decreased sarcomeric force-generating capacity (Fmax). Similarly, cardio-myocyte specific inducible BAG3 KO mice had increased ubiquitination of sarcomeric proteins that remained integrated in the lattice, reducing force generation. Importantly, BAG3 gene therapy in a mouse HF model reversed this phenotype, indicating the potential of targeting sarcomere PQC to improve contractile function. In this renewal we will address the central hypothesis that sarcomere PQC occurs via sarcomere-localized pathways and depression of these systems in HF due to BAG3 instability results in accumulation of ubiquitinated proteins that induce dysfunction. In Aim 1 we will Explore the spatiotemporal organization of the key steps in sarcomere PQC. We will use super-resolution live cell imaging in neonatal rat ventricular myocytes (NRVMs) and human iPSC-CMs to visualize the spatiotemporal interplay between BAG3, autophagosomes, lysosomes, the z-disc, and BAG3-clients at baseline and in response to various stress and stimuli, such as heat shock, localized laser damage, hypertrophic signaling, and depressed BAG3 levels. In Aim 2 we will identify the functional consequences of sarcomeric protein ubiquitination. We will use in vivo and in vitro approaches to modulate sarcomere protein ubiquitination and assess the impact on sarcomere function with biophysical assays (force- Ca2+ relationship, tension cost, in vitro motility assay, super-relaxed state, co-sedimentation). In Aim 3 we will discover the regulation of BAG3 in the cardiomyocyte and how it is altered in heart failure. We will use several transgenic mouse lines and a myocardial infarction induced heart failure model, to discover the interplay between HSP70, BAG3, heart failure, and sarcomere PQC. We expect to identify new methods to stabilize BAG3 in the failing heart as a possible therapeutic strategy. These 3 aims establish a foundational understanding of sarcomere PQC, functional consequences of its misregulation, and how it can be modulated in vivo.